The incidence of both Type 1 and Type 2 diabetes mellitus is rising to alarming rates in the United States. With significant advances in molecular biology and gene therapy in recent years, new approaches for therapy of diabetes are coming from studies of pancreatic B-cell development. One very promising approach is to force precursor cell types to develop into insulin-producing cells by use of "gene regulators," or transcription factors. The long range objective of our laboratory is to understand the mechanisms by which transcription factors direct the differentiation of the insulin-producing 13-cells within the pancreatic islets of Langerhans. Our strategy for this proposal is to focus on Nkx6. 1, a transcription factor that controls the final step of B-cell differentiation. Targeted disruption of Nkx6. 1 in mice leads to embryos that lack B-cells, with no changes in the other cell types that comprise the islets of Langerhans. Based on our preliminary data, we hypothesize that the transcriptional function of Nkx6. 1 is specifically regulated, and to control 13-cell differentiation this factor undergoes a series of intramolecular structural adjustments and intermolecular protein interactions that serves to modulate its DNA binding and transactivation functions. Our specific aims are therefore directed toward a systematic analysis of the DNA binding and transactivation properties of Nkx6. 1, in order to form a model of how this factor functions at the molecular level to cause the final differentiation of B-cells. 1. Determine how the carboxyl terminus of Nkx6. I modulates DNA binding and sequence recognition. 2. Determine how the transcriptional repression and DNA binding activities of Nkx6.1 are modified by protein-protein interactions. 3. Determine the in vivo and in vitro distribution of target genes bound by Nkx6. 1. To achieve these aims, we will utilize in vitro and in vivo assays protein and DNA interactions, cell culture models of 13-cells, and chromatin immunoprecipitation assays. The studies proposed here will provide the framework for understanding the molecular events governing differentiation in the B-cell lineage, and can eventually be applied to engineering new B-cells for patients with diabetes. .